Alzheimer's dementia (AD) is the most prevalent neurodegenerative disorder, affecting about 2-5% of the population by the age of 65 years and more than 35% by the age of 85. The disease comprises more than 75% of all dementia cases. Worldwide there are an estimated 18 million AD patients and this number is expected to double in the next 20 years. Besides therapy, early and objective diagnosis remains the major clinical problem. Diagnosis of AD is only definite and certain by post-mortem pathological analysis of the brain for the presence of extracellular deposits of beta-amyloid (Aβ) peptides, known as amyloid plaques, and intracellular aggregates of hyperphosphorylated protein tau in the form of paired helical filaments (PHF) and neurofibrillary tangles (NFT). Based on clinical examination and on cognition tests, the diagnosis evolves from mild-cognitive impairment (MCI) to possible AD and probable AD. In the late stages, trained clinicians can only diagnose AD with 80-85% certainty, leaving a high number of false positive and false negative cases. Hence, there is an urgent need for early and accurate diagnosis as this would allow for proper and effective treatment. Such treatment is not yet available, in part due to the fact that experimental drugs must be tested in early stage of AD before the brain suffers from irreversible damage (Tarditi et al., 2009). Diagnosis based on the imaging of brain, such as MRI or PET, has improved enormously during the last decade but, nonetheless, also these diagnostic systems are only accurate at the later phases of AD and therefore are not useful for the recognition of early stage AD cases (van Berckel and Scheltens, 2007). Therefore, enormous efforts are put in searching for biomarkers that could allow for an objective differentiating measurement in body fluids such as blood plasma or cerebrospinal fluid (CSF).
For the latter, Innogenetics NV (INNX) provides diagnostic kits for clinical practice based on measurement of total tau protein, phosphorylated tau protein, and the amyloid peptide Aβ42 in the form of ELISA-kits INNOTEST® hTau, INNOTEST® phospho-Tau (P-Thr181) and INNOTEST® β-amyloid(1-42), respectively, as well as the multi-parametric immunotest INNO-BIA AlzBio3. These kits give satisfactory results when it comes to discriminate AD patients from healthy persons based on CSF measurements, with an accuracy up to 83%. However, they fall short when it comes to discriminate AD, especially early stage, from other types of dementia or to measure the biomarkers in plasma samples.
To date, there are no effective therapeutic drugs available for AD and the current treatment is limited to the administration of drugs that temporarily suppress the symptoms, such as the cholinesterase inhibitor Reminyl® or the NMDA antagonist Memantine®. For therapeutic intervention, research of the last decades placed a major focus on the prevention or clearance of Aβ deposits. However, more and more data are indicating that this may not be the best approach. Not only is there a poor correlation between Aβ plaque pathology and the clinical progression of AD, but several reports demonstrated the lack of a significant clinical benefit upon immunological clearance of Aβ deposits in the brain of transgenic (Tg) mice and AD patients (Weiner and Frenkel, 2006; Josephs et al., 2008; Tarawneh and Holtzman, 2009). In contrast, the accumulation of paired helical filaments (PHF or PHFtau) and neurofibrillary tangles (NFTs) in the AD brain is highly correlated with disease progression and it is commonly used to stage AD by post-mortem histopathology (Braak and Braak, 1991). Furthermore, the suppression of protein tau in Tg mice models, either genetically or by means of immunological interference, led to reduction of the brain pathology and functional improvement (Santacruz et al., 2005; Oddo et al., 2006; Asuni et al., 2007; Sigurdsson, 2008). Hence, it appears that protein tau might be a good target for therapeutic intervention, either alone or in combination with clearance of toxic Aβ peptides. Unfortunately, there is only limited knowledge about the pathways and molecular mechanisms that drive protein tau to form PHF and NFT, neither is there a profound insight in the structure of the actual toxic tau agent(s), which could be conformer(s), oligomers, paired helical filaments (PHF) and neurofibrillary tangles (NFT).
In contrast to small proteins such as prion, synuclein and peptides such as Aβ where relatively few post-translational modifications and pathological mutations are associated with disease and oligomerization, the microtubule-associated protein tau is a challenge. Many different post-translational modifications, alternative splicing and many different mutations define a wide range of disease associations ranging from Parkinson's disease over frontotemporal lobe dementia to Alzheimer's disease. To study the biochemistry and pathogenicity of protein tau, several model systems have been developed, which include flies and worms as well as cell lines, besides Tg mice.
Yeast is a well-characterized simple system in which the cellular biology is well-described in molecular terms and which can be used to express, purify and characterize specific molecular forms of specific proteins in a timely manner. Recently, so-called humanized yeast models were developed that recapitulated important aspects of a tauopathy. These yeast models displayed tau (hyper)phosphorylation, tau conformational changes and tau self-aggregation. Importantly, creation of the major pathogenic phospho-epitopes on human tau, such as the AD2 (P-Ser396/P-Ser404) and the PG5 (P-Ser409) epitopes, were found to be modulated by Mds1 and Pho85, the yeast orthologues of the two most important mammalian tau kinases, i.e. glycogen synthase kinase 3β (GSK-3β) and cyclin-dependent kinase 5 (cdk5), respectively. Negative and positive modulation of the phosphorylation status of protein tau by expression in the MDS1 and PHO85 deletion strains, respectively, allowed to confirm that hyperphosphorylation correlated with the immunoreactivity of tau to the conformation-dependent antibody MC1 and with the amount of sarkosyl-insoluble tau (Vandebroek et al., 2005). An inverse correlation between hyperphosphorylation of tau and the ability of tau to perform its normal physiological function, i.e. to bind and stabilize microtubuli, could also be demonstrated (Vandebroek et al., 2006).
A detailed analysis of several clinical tau mutants produced in these humanized yeast models demonstrated that the mutants tau-P301L and tau-R406W were less phosphorylated at Ser409 and that this coincided with a markedly lower level of the sarkosyl-insoluble fraction, suggesting that the PG5 epitope is an important determinant for tau aggregation. This finding was substantiated by the observation that the synthetic tau-S409A mutant failed to produce significant amounts of sarcosyl-insoluble tau, while its pseudo-phosphorylated counterpart tau-S409E yielded more or comparable sarkosyl-insoluble tau as wild-type tau. It was further shown that oxidative stress and mitochondrial dysfunction strongly induced tau-insolubility independent of the phosphorylation status (Vanhelmont et al., 2010).
In addition, the humanized yeast strains also allowed to further elucidate the role of the peptidyl-prolyl cis/trans isomerase Pin1 in the pathophysiology of protein tau. Reminiscent of data recently obtained with mammalian systems (Hamdane et al., 2006), it was found that Pin1 and its yeast orthologue Ess1 lower phosphorylation of tau at Thr231 and reduce the level of the in pathologic tau-conformation detectable by MC1 (De Vos et al., International Journal of Alzheimer's Disease Volume 2011 (2011), Article ID 428970, 16 pages).
In order to specifically detect pathogenic forms of tau, several strategies have been attempted. Specific detection of (hyper)phosphorylated tau is one of the approaches developed. These antibodies only recognize their epitopes in their phosphorylated state. Examples of antibodies specific for (hyper)phosphorylated tau are AT8, specific for P-Ser202/P-Ser205 (WO 1993/008302)(Mercken et al., 1992), AT100, specific for P-Thr212/P-Ser214 (Zheng-Fischhofer et al., 1998), AT180, specific for P-Thr231/P-Ser235 (WO 1995/017429), AT270, specific for P-Thr181 (WO 1995/017429), AD2, specific for P-Ser396/P-Ser404 (Buée-Scherrer et al., 1996), and PG5, specific for P-Ser409 (Jicha et al., 1999), anti-Tau pS422 specific for P-Ser422 (WO2012/142423),
An alternative approach is the detection of pathogenic tau species with antibodies recognizing a conformational epitope of pathogenic tau. Examples of such antibodies are A1z50, whose conformational epitope encompasses the N-terminus and one or more microtubule-binding repeats of a single tau molecule (Carmel et al., 1996), and MC1 having a conformational epitope comprising amino acids 5-15 and 312-322 (Jicha et al., 1997). Conformational epitopes may be continuous or not, but typically, they are destroyed by denaturation, e.g. during SDS-PAGE.
Still another strategy has been the detection of processed tau by specific antibodies. Examples of such antibodies include mAb 423, which recognizes tau truncated at G1u391 (Novak et al., 1993), and DC11, which specifically binds to tau truncated at both the N- and C-terminal ends present in AD brains but not in normal brains (Vechterova et al., 2003).
In still a further approach, antibodies have been generated against tau-liposomal vaccines and were shown to specifically bind to phosphorylated tau peptides (WO2010/115843 and WO2012/045882).
In the case of Aβ, antibodies have been developed that preferentially recognize oligomers and/or aggregates, such as protofibrillar aggregates (WO 2004/024090; WO 2005/123775; (Kayed et al., 2010)), amylospheroids (WO 2006/016644), dimeric and higher order oligomeric Aβ (WO 2007/062088), dimers (WO 2008/084402) oligomers and fibrils (WO 2007/096076), and small soluble oligomers called Aβ-derived diffusible ligands (ADDLs; WO 2003/104437; WO 2006/014478; WO 2006/055178). Also for α-synuclein, aggregation of which in neuronal cytoplasmic inclusions known as Lewy bodies is a hallmark for Parkinson's Disease, antibodies have been disclosed that specifically detect oligomeric forms (Emadi et al., 2007; Emadi et al., 2009).
To date, despite several years of research on tau aggregation, no antibodies preferentially binding aggregated tau are available. The present invention provides tau antibodies or antibody fragments preferentially binding to phosphorylated tau aggregates, compositions comprising such antibodies or antibody fragments, nucleic acids encoding such antibodies or antibody fragments, and cell lines and hybridomas secreting such antibodies or antibody fragments. Also provided are methods to induce an immune response towards phosphorylated tau aggregates in an animal, as well as methods to obtain the antibodies or antibody fragments of the invention. The invention further provides methods and kits for the detection of aggregated tau and for the in vitro diagnosis of tauopathies using these antibodies or antibody fragments. Further provided are methods to identify compositions which interfere with formation or stability of such tau aggregates. Also provided are prophylactic or therapeutic compositions for the prevention or treatment of a tauopathy, comprising the antibody or antibody fragment of the invention.